ABSTRACT We propose that extracellular vesicles (EV) released from compartments along the endosomal-lysosomal (EL) pathway both reflect and contribute to the pathogenesis of Alzheimer's disease (AD). We further hypothesize that AD-related dysfunctions in the EL pathway causing mistrafficking or preventing efficient degradation affect exocytosis and brain EV levels. These ideas are supported by our preliminary findings showing significant changes in the levels and content of brain EV in Down syndrome and with apolipoprotein E4 (ApoE4) expression, both of which cause EL alterations. We will investigate the mechanism(s) of neuronal and glial secretion of exosomes ? the better understood EV subtype ? by late-endosome/multivesicular bodies (LE/MVB) and the impact of endosome dysfunction on exosome, and more broadly, EV biology. Important mechanistic drivers of EL pathway pathology in AD that we will examine for their impact on brain EV are the ?- site cleaved carboxyl-terminal fragment of the amyloid ? precursor protein (?CTF), hyperactivation of rab5, and altered cholesterol trafficking/metabolism. Highlighting the integrative nature of this Program across the entire EL pathway, we will examine the novel idea that lysosomal exocytosis is altered by lysosomal hydrolytic and pH impairments associated with AD-risk factors including cholesterol perturbation, ApoE4, and loss-of-function mutations in presenilin 1 and 2. Aim 1 will use genetic models and crosses (Core B) and a high cholesterol diet (P1) to test in vivo the hypothesis that factors increasing endocytic drive promote EV release from the LE/MVB, while factors promoting lysosomal dysfunction will have a greater impact on exocytic release from lysosomes. We will characterize various brain EV types and their differential origins from neurons and glia. We will determine the molecular mechanisms underlying altered EV secretion, changes in EV uptake, and alterations in EV clearance from the brain in AD-relevant pathological conditions resulting from i) altered early endosomal function (with P1) and/or ii) lysosomal dysfunction (with P2). We will examine the proteome, lipidome (with Core C) and RNA content (with P4) of EV and of the intracellular compartments contributing to EV generation to identify pathogenic changes altering exocytosis. In Aim 2, using direct manipulation of EV generation and release relevant to disease mechanisms, we will identify manipulations that normalize EV levels, testing our hypothesis that restoring EV secretion can reduce cellular pathology. As a rescue paradigm, we will test cystatin C-derived small peptides to identify molecules with established cystatin C-like protective effects that, in addition to augmenting EV secretion, can alleviate endosomal pathology, induce autophagy and lysosomal degradation, and confer protection against neuronal loss and memory deficits. Thus, this Project will define the mechanisms regulating EV generation, secretion, and their content, test our hypothesis that pathogenic alterations of the EL pathway in AD disrupt brain EV release, and demonstrate that restoring normal EV production may be an innovative therapeutic approach for reducing EL pathway pathology in AD.